Thermodynamic and Kinetic Stabilities of Al(III) Complexes with N2O3 Pentadentate Ligands

Al(III) complexes have been recently investigated for their potential use in imaging with positron emission tomography (PET) by formation of ternary complexes with the radioisotope fluorine-18 (18F). Although the derivatives of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) are the most applied chelators for [Al18F]2+ labelling and (pre)clinical PET imaging, non-macrocyclic, semi-rigid pentadentate chelators having two N- and three O-donor atoms such as RESCA1 and AMPDA-HB have been proposed with the aim to allow room temperature labelling of temperature-sensitive biomolecules. The paucity of stability data on Al(III) complexes used for PET imaging instigated a complete thermodynamic and kinetic solution study on Al(III) complexes with aminomethylpiperidine (AMP) derivatives AMPTA and AMPDA-HB and the comparison with a RESCA1-like chelator CD3A-Bn (trans-1,2-diaminocyclohexane-N-benzyl-N,N′,N′-triacetic acid). The stability constant of [Al(AMPDA-HB)] is about four orders of magnitude higher than that of [Al(AMPTA)] and [Al(CD3A-Bn)], highlighting the greater affinity of phenolates with respect to acetate O-donors. On the other hand, the kinetic inertness of the complexes, determined by following the Cu2+-mediated transmetallation reactions in the 7.5–10.5 pH range, resulted in a spontaneous and hydroxide-assisted dissociation slightly faster for [Al(AMPTA)] than for the other two complexes (t1/2 = 4.5 h for [Al(AMPTA)], 12.4 h for [Al(AMPDA-HB)], and 24.1 h for [Al(CD3A-Bn)] at pH 7.4 and 25 °C). Finally, the [AlF]2+ ternary complexes were prepared and their stability in reconstituted human serum was determined by 19F NMR experiments.


Introduction
Positron emission tomography (PET) is a diagnostic imaging technique that employs chemical tracers containing positron-emitting radionuclides. Fluorine-18 ( 18 F) is the most commonly used positron emitter, mainly due to its suitable radiochemical properties and accessibility. The favorable properties of 18 F include (1) a half-life time of 110 min; (2) its almost pure positron emission (97% β + , 3% EC); (3) its decay product, 18 O, stable isotope; (4) the low energy of the emitted positrons (0.63 MeV) and the short positron range in tissues (1-2 mm in water), allowing the capture of images with good resolution [1,2].
In most cases, fluorinated tracers contain 18 F bound to a small organic molecule, such as for [ 18 F]fluorodeoxyglucose (FDG), which stands out among the 18 F radiotracers for its superior performance [3]. However, the synthesis of fluorinated tracers requires numerous and often laborious synthetic steps, organic solvents, catalysts, and high temperatures. At the same time, 18 F is obtained as an aqueous solution by proton irradiation of [ 18  which considerably reduces the nucleophilicity of the F − ions. Long drying steps, anhydrous aprotic solvents, and high temperatures are required to increase nucleophilicity. Therefore, in the field of PET tracers, there is always a need to find new methods for the rapid and efficient introduction of 18 F in complex and temperature-sensitive biomolecules.
The coordination approach for the labelling of biomolecules with 18 F consists in the formation of a strong bond between the fluorine atom and elements such as boron, silicon, or aluminum, requiring a lower activation energy for their formation than that necessary for the formation of C-F bonds. Moreover, Si-F (540 kJ mol −1 ), B-F (766 kJ mol −1 ), and Al-F (664 kJ mol −1 ) [4] are high-energy bonds potentially highly stable for in vivo imaging applications. Furthermore, the main strength of the coordination approach lies in the possibility of carrying out a single radiolabelling reaction, ideally very fast, which can be easily automated for routine production. In addition, being able to operate in an aqueous environment, long and laborious protocols are not required for the anhydrification of the 18 F ion, further accelerating the preparation process of the radiotracer.
Very recent reviews [5][6][7][8] retrace in detail all the research carried out on aluminum fluoride-based radiotracers since the first publication in 2009 by McBride et al. [9], who were the first to explore the Al 18 F method for the radiolabelling of peptides of pharmaceutical interest. They tested several derivatives of the hexa-and pentadentate macrocyclic ligands based on 1,4,7-triazacyclononane (NOTA and NODA, respectively; see Figure 1) having N 3 O 3 and N 3 O 2 coordinative sets of donor atoms for the coordination of [Al 18 F] 2+ , respectively [10,11]. Advanced clinical studies using peptides (Alfatide I and II [12], octreotide [13], neurotensin [14]) conjugated to a macrocyclic chelator labelled with [Al 18 F] 2+ have been published in recent years for the visualization of neuroendocrine tumors, prostate cancer, and metastases in lung cancer, lymph nodes, or bones. Although these macrocyclic chelators show considerable potential, the high temperature required for the complexation reaction (≥100 • C) is still the main limit to the widespread application of this radiolabelling approach, especially for the labelling of temperature-sensitive biomolecules. Thus, Bormans and co-authors initially used EDTA-based pentadentate chelators [15], obtaining good labelling yields but low stability in physiological conditions and in serum of the [Al 18 F] 2+ complexes. To increase the kinetic inertness, the rigidity of the chelator was therefore increased using CDTA-like systems (CDTA = trans-1,2-diaminocyclohexane-N,N,N ,N -tetraacetic acid). A bifunctional chelator, called RESCA1, was then conjugated to a nanobody or to interleukin-2 and labelled with [Al 18 F] 2+ at room temperature and tested in vivo with reasonable success [16][17][18]. Our group has recently proposed the use of 2aminomethylpiperidine (AMP)-based pentadentate chelators for [Al 18 F] 2+ labelling [19]. In particular, the AMPDA-HB chelator bearing two acetate and one phenolate pendant arms and, therefore, a N 2 O 3 donor set, showed particular high labelling yields with [Al 18 F] 2+ at room temperature and 5-6.5 pH range. Moreover, the labelled complex highlighted high stability in vitro (up to 4 h in PBS, serum, and EDTA solutions) and in vivo, rapid hepatobiliary and renal excretion, and low accumulation in the bones.
Although over the last decade the [ 18 F]AlF approach has become part of the recognized procedures in the field of nuclear medicine and it is actually an effective tool in radiopharmaceutical design [5][6][7][8], there is still a scarcity of data in the literature about the thermodynamic and kinetic properties of the Al 3+ and AlF 2+ complexes. It should be highlighted that radiochemists and medical researchers usually focus on radiochemical yields and in vivo stability, but a more detailed knowledge of the physico-chemical properties of the Al(III) complexes itself is also very important before applying these systems in vivo. Only the [Al(NOTA)] and [Al(F)(NOTA)] − systems have been investigated and the thermodynamic stability constant of the Al(III) complex and its dissociation rates in acidic and alkaline solutions reported [20]. Importantly, the recently proposed non-macrocyclic pentadentate N 2 O 3 chelators for AlF 2+ complexation ( Figure 1) have been successfully labelled with [Al 18 F] 2+ and some of them tested in vivo, but no data on thermodynamic stability and kinetic inertness of the Al(III) complexes have been yet reported. Thus, in this work, we carried out a detailed characterization of the Al 3+ complexes with AMPTA, AMPDA-HB, and CD3A-Bn, a non-conjugatable analogue of RESCA1 (Figure 1), using pH potentiometry, UV spectrophotometry, and 1 H and 27 Al NMR spectroscopy. A 19 F NMR study on the mixed AlF 2+ complexes and their stability in serum is also reported. we carried out a detailed characterization of the Al 3+ complexes with AMPTA, AMPDA-HB, and CD3A-Bn, a non-conjugatable analogue of RESCA1 (Figure 1), using pH potentiometry, UV spectrophotometry, and 1 H and 27 Al NMR spectroscopy. A 19 F NMR study on the mixed AlF 2+ complexes and their stability in serum is also reported.

Figure 1.
Chelating ligands discussed in the text.

Results and Discussion
The ligands 2-AMPTA and 2-AMPDA-HB were synthesized as reported previously by our group [19,21], whereas CD3A-Bn was synthesized as reported in the literature [22]. The Al 19 F complexes were also prepared by first forming AlF 2+ , mixing AlCl3 and NaF in water, followed by the complexation with the specific ligand. Al 19 F complexes were purified by semi-preparative HPLC-MS and characterized by ESI-MS and 27 Al and 19 F NMR spectroscopy (see ESI).

Equilibrium Properties of the Al(III) -AMPTA, Al(III)-AMPDA-HB, and Al(III)-CD3A-Bn Systems
Since the thermodynamic properties of any metal complex proposed for in vivo applications must be characterized by high thermodynamic stability, the equilibrium properties of AMPTA, AMPDA-HB, and CD3A-Bn ligands and their Al(III) complexes were investigated in detail. First, the protonation constants of the ligands, defined by Equation (1), were determined by pH-potentiometry and the log-Ki H values are listed in Table 1.
The protonation sequence of AMPTA and AMPDA-HB was investigated by 1 H NMR spectroscopy and spectrophotometry, respectively [21]. According to the 1 H NMR studies, first and second protonations of AMPTA took place at the N-atoms of the piperidine moiety and of the iminodiacetic acid group. At pH < 4, further protonation of AMPTA occurs on the carboxylate groups of the iminodiacetic acid [21]. On the other hand, spectrophotometric studies revealed that the first and second protonations of the AMPDA-HB take place at the nitrogen of the aminomethyl group (the protonation occurs partially at the N-atom and the phenolate-O¯ group due to the H-bond formation) and the phenolate-O − group. The logK2 H value of AMPDA-HB is comparable with that of phenol (logK H = 10.0, 0.1 M NaClO4) [23].

Results and Discussion
The ligands 2-AMPTA and 2-AMPDA-HB were synthesized as reported previously by our group [19,21], whereas CD3A-Bn was synthesized as reported in the literature [22]. The Al 19 F complexes were also prepared by first forming AlF 2+ , mixing AlCl 3 and NaF in water, followed by the complexation with the specific ligand. Al 19 F complexes were purified by semi-preparative HPLC-MS and characterized by ESI-MS and 27 Al and 19 F NMR spectroscopy (see ESI). Since the thermodynamic properties of any metal complex proposed for in vivo applications must be characterized by high thermodynamic stability, the equilibrium properties of AMPTA, AMPDA-HB, and CD3A-Bn ligands and their Al(III) complexes were investigated in detail. First, the protonation constants of the ligands, defined by Equation (1), were determined by pH-potentiometry and the logK i H values are listed in Table 1.
The protonation sequence of AMPTA and AMPDA-HB was investigated by 1 H NMR spectroscopy and spectrophotometry, respectively [21]. According to the 1 H NMR studies, first and second protonations of AMPTA took place at the N-atoms of the piperidine moiety and of the iminodiacetic acid group. At pH < 4, further protonation of AMPTA occurs on the carboxylate groups of the iminodiacetic acid [21]. On the other hand, spectrophotometric studies revealed that the first and second protonations of the AMPDA-HB take place at the nitrogen of the aminomethyl group (the protonation occurs partially at the N-atom and the phenolate-O − group due to the H-bond formation) and the phenolate-O − group. The logK 2 H value of AMPDA-HB is comparable with that of phenol (logK H = 10.0, 0.1 M NaClO 4 ) [23]. Further protonation of AMPDA-HB takes place at the non-protonated piperidine-N and the carboxylate-O donor atoms in the pendant arms [21]. − instead of Cl − has practically no effect for the protonation constants of these pentadentate ligands. The stability and protonation constants of the Al(III) complexes of AMPTA, AMPDA-HB, and CD3A-Bn, defined by Equations (2)-(4), were investigated by pH-potentiometry and by multinuclear NMR spectroscopy at 25 • C in 0.15 M NaNO 3 solution. (2) In order to avoid the hydrolysis of the Al(III) ion, the pH-potentiometric titration of the pre-prepared complexes of AMPTA, AMPDA-HB, and CD3A-Bn ligands was performed at pH  (3) and (4). In calculating the equilibrium constants, the best fitting of the mL NaOH-pH data was obtained by assuming the formation of AlL and AlLH −1 species.
To determine the stability constant of the Al(III) complexes, the 1 H and 27 Al NMR spectra of the Al 3+ -AMPTA, Al 3+ -AMPDA-HB, and Al 3+ -CD3A-Bn systems were recorded in the pH range 8.0-12. 1 H and 27 Al NMR spectra of the three systems are shown in Figures [28,29]. As shown in Table 1 Table 1). In the Al(III) complexes of the present work, the Al(III) ion is presumably coordinated by 2 amino-N, two or three carboxylate-O − , and the very basic phenolate-O − donor atoms, whereas the sixth coordination site of the Al(III) ion is occupied by the inner-sphere water molecule to complete the octahedral coordination geometry [30]. Interestingly, the logK AlLH     The species distribution diagrams and the 1 H and 27 Al NMR spectra (Figures 2-4 and S1-S6) indicate that formation of the three Al(III) complexes is completed at pH > 4.5. In     The Al(III) complexes are generally characterized by relatively low thermodynamic stability and high kinetic inertness due to the slow ligand exchange reactions. However, the large excess of the endogenous competition partners (mainly Cu(II) and Zn(II) and/or transferrin) [33] Figure 5.
see Figures S2, S4 and S6) and of the 1 H NMR signals of the free AMPTA, AMPDA-HB, and CD3A-Bn ligands ( Figures S1, S3 and S5). The intensity of the 27 Al NMR signal of [Al(OH)4] − increases with increasing pH due to the dissociation of [AlLH −1 ] species in the pH range 8.0-11.0. Interestingly, the 1 H NMR signal of the free AMPTA, AMPDA-HB, and CD3A-Bn ligands has a significant upfield shift due to the deprotonation of the free ligands at pH > 9.0.

Kinetic Inertness of the [Al(AMPTA)], [Al(AMPDA-HB)], and [Al(CD3A-Bn)]
The Al(III) complexes are generally characterized by relatively low thermodynamic stability and high kinetic inertness due to the slow ligand exchange reactions. However, the large excess of the endogenous competition partners (mainly Cu(II) and Zn(II) and/or transferrin) [33]   The rates of the metal exchange reactions were studied in the presence of large Cu(II)citrate excess, so the transmetallation can be treated as a pseudo-first-order kinetic process and the reaction rates can be expressed by Equation (6): where k d is a pseudo-first-order rate constant and [AlL] t is the total concentration of the Al(III) complexes at the time t, respectively.  Figure 6.
where kd is a pseudo-first-order rate constant and [AlL]t is the total concentration of the Al(III) complexes at the time t, respectively. The pseudo-first-order rate constants characterizing the transmetallation reactions of [Al(AMPTA)], [Al(AMPDA-HB)], and [Al(CD3A-Bn)] with Cu(II) at different pH values in the presence of citrate are shown in Figure 6.    By taking into account all possible pathways, the rate of the dissociation of the Al(III) complexes can be expressed by Equation (11).  (8)), the k d pseudo-first-order rate constants presented in Figure 6 can be expressed by Equation (12):  Table 2 and compared with those of [Al(NOTA)].   In the fitting procedure of the kinetic data obtained for the [Al(AMPTA)]-Cu(II)-citrate and [Al(AMPDA-HB)]-Cu(II)-citrate reacting system, the first term of the numerator in Equation (12) was neglected due to the relatively fast OH − assisted dissociation (k Al(L)H −2 and k Al(L)H −3 ; see Equations (9) and (10)

Serum Stability of AlF 2+ -Complexes
The Al 19 F complexes were dissolved in an aqueous solution of Seronorm ® and 19 F NMR spectra were recorded at different times in order to determine the stability of the [Al(F)(L)] − ternary complexes. The spectra were recorded every 15 min for the first 3 h and then after 24 h (Figures S8 and S9). No substantial variation in the 19 F NMR signals' chemical shift and intensity was observed in 24 h, thus highlighting the excellent stability of the aluminum fluoride ternary complexes in physiological conditions.

General
All chemicals were purchased from Sigma-Aldrich or Alfa Aesar unless otherwise stated and were used without further purification. The 1 H and 13 C NMR spectra were recorded using a Bruker Advance III 500 MHz (11.4 T) spectrometer equipped with 5 mm PABBO probes and BVT-3000 temperature control unit. Chemical shifts δ are reported relative to TMS and were referenced using the residual proton solvent resonances. HPLC analyses and mass spectra were performed on a Waters HPLC-MS system equipped with a Waters 1525 binary pump. Analytical measurements were carried out on a Waters XTerra MS C18 (5 µm 4.6 × 100 mm) and on a Waters C18 XTerra Prep (5 µm 19 × 50 mm) for preparative purposes. Electrospray ionization mass spectra (ESI MS) were recorded using an SQD 3100 Mass Detector (Waters), operating in positive or negative ion mode, with 1% v/v formic acid in methanol as the carrier solvent.

Equilibrium Measurements
The chemicals used for the experiments were of the highest analytical grade. The concentration of the ZnCl 2 and CuCl 2 solutions was determined by complexometric titration with standardized Na 2 H 2 EDTA and xylenol orange (ZnCl 2 ) and murexid (CuCl 2 ) as indicators. Al(NO 3 ) 3 was prepared by dissolving metallic aluminum (99.9%, Fluka) in 6 M HNO 3  The waiting time between two pH measurements was 60 s. For the equilibrium calculations, the stoichiometric water ionic product (pK w ) was also needed to calculate [OH − ] values under basic conditions. The V NaOH -pH read data pairs of the HNO 3 NaOH titration obtained in the pH range 10.5-12.0 were used to calculate the pK w value (pK w = 13.78). The protonation and stability constants were calculated with the PSEQUAD program [35].

NMR Experiments
1 H, 19 F, and 27 Al NMR measurements were performed using either a Bruker Avance III (9.4 T) spectrometer, equipped with Bruker Variable Temperature Unit (BVT) and Bruker Cooling Unit (BCU), or a BB inverse z gradient probe (5 mm 6 ] 3+ for 27 Al as the external standard.

Kinetic Studies
The rates of the exchange reactions taking place between [Al(AMPTA)], [Al(AMPDA-HB)], and [Al(CD3A-Bn)] and Cu(II) in the presence of citrate were studied by spectrophotometry, following the formation of the resulting Cu(II) complexes at 295 nm, with the use of 1.0 cm cells and a PerkinElmer Lambda 365 UV-Vis spectrophotometer at 25 • C in 0.15 M NaCl solution. The concentration of the Al(III) complexes was 0.1 mM, while that of Cu(II) was 10 times higher, to ensure pseudo-first-order conditions. In order to prevent the hydrolysis of Al(III) and Cu(II) ions, the transmetallation reactions were studied in the presence of citrate excess ([Cit] t = 10 and 20 mM). The exchange rates were studied in the pH range 7.0-10.5. For keeping the pH values constant, HEPES and piperazine buffer (0.01 M) were used. The pseudo-first-order rate constants (k d ) were calculated by fitting the absorbance data to Equation (13).
where A t , A 0 , and A p are the absorbance values at time t, the start of the reaction, and at equilibrium, respectively. The calculation of the kinetic parameters was performed by the fitting of the absorbance-time and relaxation rate-time data pairs with the Micromath Scientist computer program (version 2.0, Salt Lake City, UT, USA).

Conclusions
The semi-rigid pentadentate chelators investigated in this work are among the most promising systems for room temperature [Al 18 F] 2+ labeling of biomolecules for PET imaging, and thus, the thermodynamic stability and kinetic inertness of their Al(III) complexes were studied in detail. In particular, [Al(AMPDA-HB)] showed the highest thermodynamic stability constant with a logK AlL of 18.6, about four orders of magnitude higher than that of [Al(AMPTA)] and [Al(CD3A-Bn)]. With regards to the kinetic inertness, the dissociation half-life of [Al(CD3A-Bn)] is about two and five times higher than [Al(AMPDA-HB)] and [Al(AMPTA)] due to the slower OH − -assisted dissociation of the hydroxo-complex ([Al(L)H −1 ] −1 ) at pH 7.4. Moreover, the Al 19 F complexes are shown to be stable with no change in the 19 F NMR peak in human serum for at least 24 h. All these data confirm that the [Al 18 F] 2+ -labelled pentadentate ligands discussed in this work are well suitable for in vivo PET imaging once conjugated to a biomolecule, with [Al(AMPDA-HB)] showing a much better thermodynamic stability and [Al(CD3A-Bn)] a two-fold higher kinetic inertness. Thus, the conjugation of these pentadentate chelators to selected biomolecules will bring important innovation in the field of AlF-18 radiolabelling, providing a new tool for oncological or immunoPET imaging. Clearly, the in vivo application is essential to determine the real applicability of these chelators with the AlF-18 approach, but the results reported in this work allow for defining the chemical safety of these systems.